DESCRIPTION: Actinobacillus actinomycetemcomitans is a facultative anaerobe that has been implicated in a variety of periodontal diseases. Its presence is most closely associated with localized juvenile periodontitis (LJP) where it is believed to be the etiologic agent. The role this organism plays in other periodontal diseases, however, is not entirely clear. While A. actinomycetemcomitans produces a number of virulence factors, a leukotoxin (Ltx) appears to be most associated with enabling this organism to establish an ecological niche and thereby achieve its pathogenic potential. This concept is supported by an increasing number of clinical studies that demonstrate a reproducible ability to culture strains with the high leukotoxic phenotype from patients with active LJP. The mechanism by which A. actinomycetemcomitans Ltx kills target cells is ambiguous since two mechanisms of eukaryotic cell death have been identified: necrosis and apoptosis. High concentrations (> 104 M) of Ltx induce very rapid target cell death with little morphologic or cytometric evidence of apoptosis. Nevertheless, cells exposed to low concentrations of Ltx exhibit an alteration in mitochondrial transmembrane permeability that is followed by: 1) dissipation of the mitochondrial transmembrane potential, 2) release of apoptosis-inducing factors, 3) generation of reactive-oxygen radicals, and 4) depletion of ATP stores. The earliest change that we have observed following the addition of Ltx to susceptible cells is a rise in intracellular calcium [Ca2+]; which is both immediate (within 10 sec) and sustained. The Ltx-induced Ca 2+ influx and apoptosis exhibit an identical dose-dependence and point to a mechanistic connection linking of Ca 2+ influx and the onset of apoptosis. The ability of a mAb to neutralize the cytolytic activity of the Ltx and to inhibit the [Ca 2+]; changes are a strong indication that the reaction begins with Ltx. The proposed investigations are designed to increase our understanding of Ltx cytolysis by: 1) exploring early steps that result in Ltx-induced [Ca2+] c increases in target cells; 2) identifying the origin(s) of the increased [Ca2+] c seen in Ltx-treated cells; 3) defining effects Ltx-induced [Ca 2+] c increases have on cellular organelles and 4) characterizing the function of the endoplasmic reticulum in Ltx-treated cells.